The glycolytic enzyme, phosphoglycerate mutase, has critical roles in stomatal movement, vegetative growth, and pollen production in Arabidopsis thaliana.

Stomatal movements require massive changes in guard cell osmotic content, and both stomatal opening and stomatal closure have been shown to be energy-requiring processes. A possible role for glycolysis in contributing to the energetic, reducing requirements, or signalling processes regulating stomatal movements has not been investigated previously. Glycolysis, oxidization of glucose to pyruvate, is a central metabolic pathway and yields a net gain of 2 ATP and 2 NADH. 2,3-biphosphoglycerate-independent phosphoglycerate mutase (iPGAM) is a key enzymatic activity in glycolysis and catalyses the reversible interconversion of 3-phosphoglycerate to 2-phosphoglycerate. To investigate functions of iPGAMs and glycolysis in stomatal function and plant growth, Arabidopsis insertional mutants in At1g09780 and At3g08590, both of which have been annotated as iPGAMs on the basis of sequence homology, were identified and characterized. While single mutants were indistinguishable from the wild type in all plant phenotypes assayed, double mutants had no detectable iPGAM activity and showed defects in blue light-, abscisic acid-, and low CO(2)-regulated stomatal movements. Vegetative plant growth was severely impaired in the double mutants and pollen was not produced. The data demonstrate that iPGAMs and glycolytic activity are critical for guard cell function and fertility in Arabidopsis.

Predicting the success of fragment screening by X-ray crystallography.

Fragment screening using X-ray crystallography is a method that can provide direct three-dimensional readouts of the structures of protein-small molecule complexes for lead development and fragment-based drug discovery. With current technology, an amenable crystal form can be screened crystallographically against a library of 1000-2000 fragments in 1-2 weeks. We have performed over a dozen crystallographic screening campaigns using our own compound collection called Fragments of Life™ (FOL). While the majority of our fragment screening campaigns have generated multiple hits, some unexpectedly turned out to be nonproductive, either yielding no bound ligands, or only those thought to be inadequate for lead development. In this chapter, we have attempted to identify one or more parameters which could be used to predict whether a crystallized protein target would be a good candidate for fragment hit discovery. Here, we describe the parameters of crystals from 18 fragment screening campaigns, including six unsuccessful targets. From this analysis, we have concluded that there are no parameters that are absolutely predictive of fragment screening success. However, we do describe a parameter we have termed pocket factor which provides a statistically significant variance between nonproductive targets and productive targets shown to bind fragments. The pocket factor is calculated using a novel method of consensus scoring from three distinct pocket-finding algorithms, and the results may be used to prioritize targets for fragment screening campaigns based on an initial crystal structure.

Crystal structure of human bisphosphoglycerate mutase.

Bisphosphoglycerate mutase is a trifunctional enzyme of which the main function is to synthesize 2,3-bisphosphoglycerate, the allosteric effector of hemoglobin. The gene coding for bisphosphoglycerate mutase from the human cDNA library was cloned and expressed in Escherichia coli. The protein crystals were obtained and diffract to 2.5 A and produced the first crystal structure of bisphosphoglycerate mutase. The model was refined to a crystallographic R-factor of 0.200 and R(free) of 0.266 with excellent stereochemistry. The enzyme remains a dimer in the crystal. The overall structure of the enzyme resembles that of the cofactor-dependent phosphoglycerate mutase except the regions of 13-21, 98-117, 127-151, and the C-terminal tail. The conformational changes in the backbone and the side chains of some residues reveal the structural basis for the different activities between phosphoglycerate mutase and bisphosphoglycerate mutase. The bisphosphoglycerate mutase-specific residue Gly-14 may cause the most important conformational changes, which makes the side chain of Glu-13 orient toward the active site. The positions of Glu-13 and Phe-22 prevent 2,3-bisphosphoglycerate from binding in the way proposed previously. In addition, the side chain of Glu-13 would affect the Glu-89 protonation ability responsible for the low mutase activity. Other structural variations, which could be connected with functional differences, are also discussed.

Suppressors of T-cell receptor signaling Sts-1 and Sts-2 bind to Cbl and inhibit endocytosis of receptor tyrosine kinases.

The ubiquitin (Ub) ligase Cbl plays a critical role in attenuation of receptor tyrosine kinase (RTK) signaling by inducing ubiquitination of RTKs and promoting their sorting for endosomal degradation. Herein, we describe the identification of two novel Cbl-interacting proteins, p70 and Clip4 (recently assigned the names Sts-1 and Sts-2, respectively), that inhibit endocytosis of epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor. Sts-1 and Sts-2 contain SH3 domains that interacted with Cbl, Ub-associated domains, which bound directly to mono-Ub or to the EGFR/Ub chimera as well as phosphoglycerate mutase domains that mediated oligomerization of Sts-1/2. Ligand-induced recruitment of Sts-1/Sts-2 into activated EGFR complexes led to inhibition of receptor internalization, reduction in the number of EGFR-containing endocytic vesicles, and subsequent block of receptor degradation followed by prolonged activation of mitogenic signaling pathways. On the other hand, interference with Sts-1/Sts-2 functions diminished ligand-induced receptor degradation, cell proliferation, and oncogenic transformation in cultured fibroblasts. We suggest that Sts-1 and Sts-2 represent a novel class of Ub-binding proteins that regulate RTK endocytosis and control growth factor-induced cellular functions.

Functional maps of the junctions between interglobular contacts and active sites in glycolytic enzymes -- a comparative analysis of the biochemical and structural data.

BACKGROUND: Oligomers and separate subunits of the glycolytic enzymes often have different catalytic properties. However, spectral data show an apparent lack of significant conformational changes during oligomerization. Since the conformation of an enzyme determines its catalytic properties, the structural mechanism(s) influencing the activity is of considerable interest. MATERIAL/METHODS: Analysis of the spatial structures of the junctions between interglobular contacts and binding sites may give a clue to the mechanism(s) of the activation. In this work, the problem was studied using available structural and biochemical data for the oligomeric enzymes of glycolysis. RESULTS: Computational analysis of the structures of the junctions has identified three structurally distinct types of junctions: 1. interglobular binding site (2 of 8 enzymes); 2. domain-domain stabilization (5 of 8); and 3. 'sequence overlap' or a local conformational change (all enzymes). Thus the catalytic activity may be influenced through the shifts of the modules of protein structure (types 1, 2) and/or due to a slight change in the local structure (type 3). The more common junctions of types 2 and 3 are well conserved among eukaryotic enzymes, which suggests their biological importance. CONCLUSIONS: The results suggest that a profound and a complex change in conformation in subunits of an oligomeric enzyme may not be necessary for a significant change in the catalytic properties. The analysis maps the residues important for the junctions and thus for the link between the catalytic activity and the oligomeric state of the enzymes.

Activating oligomerization as intermediate level of signal transduction: analysis of protein-protein contacts and active sites in several glycolytic enzymes.

A number of enzymes have inactive monomeric and active oligomeric forms. This suggests presence of definite interglobular contact -active site interaction in the enzymes. Although the phenomenon is widely studied in vitro as part of folding process the biological roles of the phenomenon, termed here as "activating oligomerization" are not clearly understood. In this work a procedure for analysis of protein-protein interactions was elaborated. Using spatial structures of several glycolytic enzymes potential role of kinase phosphorylation in regulation of oligomerization of the proteins as well as association of domains in a two-domain protein was assessed. In the enzymes 15-75% of kinase sites (mainly protein kinase C and casein kinase 2 sites) are placed in interglobular contact region(s). Upon being phosphorylated these sites may prevent oligomer formation. In structures of all the enzymes definite evidences of connection between active site and interglobular contact were found. Two structural mechanisms of interglobular contact influence on the active site were proposed. In addition to known mechanism of oligomerization initiated by allosteric metabolites the influence may be also exerted through functional sequence overlap and/or interdomain contact stabilization mechanisms. Implications for regulation of enzyme cellular function(s), signal transduction and metabolic analysis are considered. It is concluded that activating oligomerization may represent an intermediate level of enzyme cellular regulation.